Sun protection factor, total phenol, flavonoid contents and antioxidant activity of medicinal plants from Iran

Purpose: To evaluate the correlation between sun protection factor (SPF) and the content of phenol and flavonoid and antioxidant activity. Methods: Different parts of 9 medicinal plants were extracted with methanol using three extraction methods (percolation, Soxhlet and ultrasonically assisted extraction) to obtain 42 crude extracts. Their phenol and flavonoid contents, and antioxidant activities were determined using Folin-Ciocalteu reagent, aluminum chloride method and DPPH radical-scavenging activity, respectively. The SPF values were determined and correlated with the phenol and flavonoid contents as well as antioxidant activities. Results: The phenol and flavonoid contents, and antioxidant activities ranged from 54.16-688.97mg GAE/g, 13.38-146.60 mg QE/g and 9.5-1472.4 μg/mL, respectively while the SPFs were between 0.067 and 0.841. The highest SPF was related to Cucumis melo L. ultrasonically assisted leaf extract (0.841) and Artemisia absinthium L., aerial parts extracted with percolation method (0.717). A significant correlation was found between SPF and phenolic (p= 0.003) and flavonoid contents (p= 0.023). Conclusion: This study showed a correlation between SPF and phenolic and flavonoid contents. Ultrasonically assisted extract of C. melo leaf has suitable SPF and can be used in sun screen formulations.


INTRODUCTION
About one million people are diagnosed with skin cancer every year [1]. Exposure to solar radiation has negative effects on the skin [2]. Ultraviolet light is mainly responsible for skin damage [1]. Sunscreens absorb or block UV rays of sunlight. There is some information regarding possible adverse reaction by synthetic sun-screen products [3]. There is little published data describing the use of herbal sunscreens and their sun protection factor (SPF) [4].
Natural substances have been recently considered as potential sources for agents with sunscreen properties [5] because of their absorption in the UV region [6] and their antioxidant activity [7]. Green tea polyphenols, aromatic compounds from lichens, glycosides of aesculin and Murraya koenigii are examples of natural substances evaluated for their sunscreen properties [8][9][10]. UV radiation increases oxidative stress in skin cells leading to cancer initiation and promotion. There has been an increasing interest in the use of antioxidants in sunscreens to provide supplemental photo protective action. Antioxidants from natural sources may provide new possibilities for the treatment and prevention of UV mediated diseases [7].
The Iranian flora is rich in medicinal plants with a high potential for providing these antioxidants. Among them, Sambucus ebulus, Artemisia absinthium and Cucumis melo are three medicinal plants with high antioxidant activities [11][12][13]. There are very limited reports on correlation between phenol/flavonoid contents or antioxidant activity and their SPF values [14].  (Boraginaceae), petals were obtained in spring and summer, from Sari, Iran. Samples were authenticated by Dr Bahman Eslami and their voucher specimens were deposited (no. 1017-1025) in Sari School of Pharmacy herbarium. The plant materials were allowed to dry at room temperature for 2 weeks, and milled to obtain 2-3 mm particles for incubation with methanol to obtain methanolic extracts. Extracts were separated from the sample residues by filtration using filter paper and the process was repeated three times. The resulting extracts were concentrated using a rotary evaporator under vacuum at 36 ± 2 °C and the crude solid extracts were freeze-dried for complete solvent removal.

Ultrasonically-assisted extraction
Samples were extracted with methanol in an ultrasonic bath by indirect sonication (frequency of 60 kHz) at 25 ± 2 °C for 1 h. The extracts were then separated from the samples residue by filtration. Resultant extracts were concentrated in a rotary evaporator until a crude solid extracts were obtained, which then freeze-dried for complete solvent removal [15].

Soxhlet-assisted extraction
Powdered samples were incubated with methanol for 24h in a Soxhlet extractor. The extracts were concentrated in a rotary evaporator to completely remove the solvent.

Determination of total phenolic and flavonoid contents
Total phenolic content was measured colorimetrically using Folin-Ciocalteu reagent [15]. The results were expressed as gallic acid equivalents (GAE). Total flavonoids were estimated using AlCl 3 method [16] and the contents calculated based on quercetin as standard from a calibration curve.

Evaluation of DPPH radical-scavenging activity
1,1-Diphenyl-2-picryl hydrazyl radical (DPPH) was used to assay free radical-scavenging activity of the extracts [17]. To 1ml of each extract (different concentrations, ranging from 25-800 g/mL) 1ml methanol solution of DPPH (100 M) was added. After 15 min incubation at room temperature in the dark, the absorbance was recorded at 517 nm. The experiment was done in triplicates using vitamin C and butylated hydroxyl anisole (BHA) as positive controls.

Computation of in vitro SPF
The extracts were dissolved in methanol and scanned in the range of 337.5 to 292.5 nm (at five nm intervals) as previously described [18]. Screening of sun protection activity was then measured by determination of in vitro SPF based on the following equation proposed by Gharavi et al [19]: where T(λ) is the measured sun screen transmittance at λ; E(λ) is the spectral irradiance of terrestrial sunlight at λ; and ε(λ) is the erythemal action spectrum at λ. The E(λ) and ε(λ) values were calculated as described previously [19]. For each extract, T(λ) was measured three times and the means was used for SPF calculation. At least five different concentrations of each extract were used to obtain the standard curve and in calculating SPF in 2 mg/mL solution.

RESULTS
The antioxidant activity of extracts calculated using DPPH method is presented in Table 1.
Best result was that of S. ebulus flower extract obtained by percolation method (IC 50 = 9.5 ± 0.9 µg/mL). S. ebulus flower extract (prepared using Soxhlet extractor, IC 50 = 9.8 ± 0.4 µg/mL), O. orientalis aerial part extract (prepared using ultra sonication, (IC 50 = 17.9 ± 0.8 µg/mL), A. julibrissin leaf extract (prepared using ultra sonication, IC 50 = 21.6 ± 0.6 µg/mL -1 ) and S. nigra leaf extract (prepared using ultra sonication, IC 50 = 21.6 ± 0.8 µg/mL) showed satisfactory degrees of antioxidant activity. These extracts all had a good antioxidant activity compared to the positive controls, vitamin C (IC 50 = 3.7 ± 0.1 µg/mL) and BHA (IC 50 = 29.3 ± 5.9 µg/mL). The values of total phenolics and flavonoids content in the extracts are shown in Table 1. The highest value of phenolic compounds was that A. julibrissin leaf extract prepared by soxhlet extractor (688.97 ± 23.3 mg GAE/g of extract). A. julibrissin leaf extract prepared using ultra sonication also had high content of phenolic compounds as well (565.52 ± 22.8 mg GAE/g of extract). However, the highest flavonoids content (146.60 ± 5.5 mg QE/g of extract) was that of D. racemosa leaf extract prepared by percolation method. A. julibrissin leaf extract (prepared by ultrasonication, 139.66 ± 6.2 mg QE/g of extract) had considerable quantity of flavonoid compounds as well. For the SPFs values of the extracts, the highest values was for Cucumis melo leaf extract (obtained by ultra sonication) and Artemisia absinthium shoots (methanolic extract) with SPF values of 0.841 and 0.717, respectively. The correlation between SPF with either phenolic (r = 0.469, p = 0.003) and flavonoid (r= 0.355, p= 0.023) contents and antioxidant activity was significant. But no significant correlations existed between SPF and antioxidant activity (r = 0.242, p = 0.132) in the extracts (Figure 1).

DISCUSSION
In this research, 42 extracts from nine known medicinal plants were evaluated for their SPF using UV spectrophotometry. We found that S. ebulus flower extract (obtained by percolation), S. ebulus flower extract (obtained by soxhlet extractor), and O. orientalis shoot extract (obtained by ultra-sonication) had considerable antioxidant activity when compared with vitamin C and butylated hydroxyl anisole (BHA).
Antioxidant activity is important in UV protection [22]. It seems that the already reported photoprotective effect of p. umbellata is due to its antioxidant activity [23]. The flavonoids and polyphenols studied are good candidates for use in photoprotective products [24,25]. Phenolic compounds are believed to be capable of acting in redox-sensitive signaling cascades to inhibit DNA damage [23,26]. They may also be beneficial in preventing UV-induced oxygen free radical generation and lipid peroxidation.
We found that A. julibrissin and D. racemosa leaf extracts have high quantities of phenolic compounds. The highest values were obtained by ultrasonic extract of C. melo leaf and percolation extract of A. absinthium aerial parts with SPF values of 0.841 and SPF = 0.717 respectively. High SPF values have been reported from leaf extract of D. moldavica and flowering tops of V. tricolor (24.79 and 25.69, respectively) [21] often considered to be due to high phenolic contents [20]. The protective power identified in this study is vital as UV is highly genotoxic agent whose deleterious effects on human skin at DNA level has been widely accepted [22]. To prevent UV-mediated DNA damage, sun protection factors are of highly interests. Although UV radiation has some benefits, e.g., for the biosynthesis of vitamin D, its negative impact on human health is much more. Skin cancer is one of the serious results.

CONCLUSION
There is a relatively significant correlation between SPF and both phenolic and flavonoid contents but not antioxidant activity. The application of ultrasonically assisted extract may be useful in achieving high SPF values in some plants.